The present invention relates to a transformed plant having an enhanced free amino acid content, a plant having enhanced nitrogen content, a plant tolerant to nitrogen deficiency, and a method for producing the plant.
Plants are autotrophic, and they are capable of synthesizing all the compounds indispensable for living. Amino acids are included in these compounds. Plants synthesize all of the 20 naturally occurring amino acids from water, carbon dioxide and inorganic nitrogen sources utilizable in the environment by making use of light energy. Animals including human beings cannot synthesize all the amino acids. Amino acids that cannot be synthesized by animals are called “essential amino acid(s)” and the intake of the essential amino acid(s) is nutritionally important. Animals principally depend on the amino acids synthesized by the plants for these essential amino acids. Therefore, it has been considered that the improvement in the quality and amount of amino acids contained in plants is an important issue for increasing the nutritional value of plants.
Further, the increase in the capacity of plants for synthesizing amino acids is also significant from the viewpoint of the growth of the plants themselves. As described above, plants synthesize amino acids from inorganic nitrogen in the environment. For the plants, the process for the synthesis of amino acids is also the process for the assimilation and absorption of nitrogen as amino acids. Namely, plants assimilate nitrogen in its final form as ammonia into glutamic acid, and glutamic acid is distributed and utilized as a nitrogen source of various components, such as other amino acids and nucleic acid, of living bodies. Accordingly, the increase in the capacity of plants for synthesizing amino acids is, in other words, an improvement in nitrogen utilization efficiency of plants. Nitrogen is one of major limiting factors for the growth of plants. If the capacity of nitrogen assimilation may be increased as a result of the increase in the amino acid-synthesizing capacity, the acceleration of the growth of the plants is expected and, accordingly, an increase in the yield thereof is also expected. In addition, if nitrogen may be efficiently used, it would be expected that the amount of inorganic nitrogen used as fertilizers may be minimized and the environmental load may be reduced.
It is considered that one of the methods for increasing an amino acid content of plants is the enhancement of the enzymatic reaction responsible for the above-described nitrogen assimilation. Generally in plants, nitrogen is reduced to form ammonia and then assimilated into glutamic acid by glutamate synthase (GOGAT) and glutamine synthetase (GS). It has been attempted, therefore, to increase an amino acid content and nitrogen content of plants by enhancing enzyme systems concerning the process for the generation and transportation of ammonia, the regulation of GOGAT or GS activity or the transportation and translocation of the assimilated amino acids. The attempts include, for examples, the introduction of soybean GS into Lotus corniculatus (Planta 1997; 201(4): 424-33, Vincent R et al.) and the introduction of asparagine synthase derived from Arabidopsis thaliana into Arabidopsis thaliana (JP-Kokai No. 9-503389, Coruzzi G et al.). However, in such examples, a significant increase in the absolute amino acid content or the absolute nitrogen content was not found, although a change in the relative contents of amino acids was found.
Besides these investigations, an investigation has been made on a DNA binding protein which specifically binds to the promoter domain of C4 type phosphoenol pyruvate carboxylase (PEPC) gene concerning the carbon dioxide assimilation. As such a DNA binding protein, Dof1 protein and the gene thereof were found in maize (Yanagisawa, S. (1993), Trends in Plant Sci., 1 (7), 213). Dof1 is a DNA binding protein unique to plants which has only one zinc(Zn)-finger-like domain. During the investigations, using a transient expression system of maize mesophyll protoplast, it was elucidated that Dof1 accelerates the transcription of C4 phosphoenol pyruvate carboxylase (PEPC) gene. It was also elucidated that Dof1 also accelerates the transcription of C3 phosphoenol pyruvate carboxylase gene and cytoplasmic pyruvate ortho-phosphate dikinase (PPDK) gene. It was also shown that a high Dof1 activity is observed in the leaves under the light. The function of the phosphoenol pyruvate carboxylase is to assimilate bicarbonate into phosphoenolpyruvate and to provide TCA cycle with oxaloacetate, and the function of the cytoplasmic pyruvate ortho-phosphate dikinase is to generate phosphoenolpyruvate from pyruvic acid and to provide the substrate of phosphoenol pyruvate carboxylase.
The DNA binding proteins having only one zinc-finger-like domain, which derive from plants, are called “Dof family”. After the search on the genome of Arabidopsis thaliana, 37 proteins belonging to the Dof family were found. Among them, a transformed plant expressing the Dof family binding protein called OBP3 has been produced. However, among those Dof family proteins, a homology is scarcely recognized except for the Zinc-finger portion and, in addition, the functions of them have never been reported except for Dof1. In fact, no phenotypes relevant to OBP3 were reported in the transformed plants to which the above-described OBP3 gene had been introduced. Further, even for Dof1, the function of which has been elucidated to some extent, it is never reported that a transformed plant was produced into which this gene had been introduced, and the physiological function of Dof1 in the plant body has not been sufficiently elucidated. In addition, the amino acid content of the transformed plant having Dof1 gene was not determined.